Introduction
Candidatus (Ca.) Neoehrlichia (N.) mikurensis is a tick-borne pathogen found in Europe and Asia [
1], which was first reported to be a human pathogen in 2010 [
2‐
4]. It can give rise to a severe infectious disease named neoehrlichiosis that features fever and vascular events in immunocompromised patients [
5]. Immunocompetent individuals infected by
Ca. N. mikurensis may present with fever and symptoms indicative of systemic infection, isolated erythematous skin rashes, or no symptoms at all [
2,
4,
6‐
9].
Like all members of the
Anaplasmataceae family,
Ca. N. mikurensis is a strict intracellular pathogen, and consequently does not grow in cell-free media, which is why it escapes detection by routine blood cultures [
1]. At present, the only microbiological diagnostic option is PCR since there are no serological assays available. The restricted diagnostic alternatives, together with the novelty of this emerging pathogen, explain why many patients with severe neoehrlichiosis remain undiagnosed and fall under the epithet of “fever of unknown origin” [
5]. In central and northern Europe,
A. phagocytophilum serology is part of the diagnostic work-up of (tick-exposed) patients with unexplained fever. There are at least three case reports of immunocompetent patients infected by
Ca. N. mikurensis who were seropositive for
A. phagocytophilum as determined by indirect immunofluorescence assay (IFA): two of the cases had de novo production of
Anaplasma-reactive antibodies, whereas the third one had pre-existing antibodies [
2,
6]. This might indicate that
Ca. N. mikurensis infection can trigger the production of
Anaplasma cross-reactive antibodies or the occurrence of double infections with
Ca. N. mikurensis and
A. phagocytophilum. Consequently, neoehrlichiosis patients may be wrongly diagnosed with anaplasmosis. The aims of this study were to address these issues. Specifically, the goals were to investigate (1) the prevalence of
Anaplasma seropositivity among patients diagnosed with neoehrlichiosis, (2) whether neoehrlichiosis patients are misdiagnosed as anaplasmosis patients based on serological findings, and (3) the existence of
A. phagocytophilum and
Ca. N. mikurensis co-infections.
Discussion
The main finding of this study is that every fifth of the neoehrlichiosis patients had low titers of A. phagocytophilum antibodies in the blood at the time of diagnosis. This is a surprisingly high figure in view of the fact that the majority of the patients were immunocompromised. This seroreactivity to A. phagocytophilum might represent (1) previous exposure to or infection with A. phagocytophilum, (2) co-infection with Ca. N. mikurensis and A. phagocytophilum, or (3) Anaplasma-crossreactive antibodies elicited or boosted by Ca. N. mikurensis infection.
Estimates of the seroprevalence of
A. phagocytophilum antibodies in the general population in Scandinavia vary greatly. Two older studies report seroprevalence figures of 2.0 and 2.5% among Danish and Norwegian blood donors [
15,
16], but a newer Norwegian study gives a figure of 16% [
17]. The estimates of the
A. phagocytophilum seroprevalence in populations that are heavily tick-exposed range from 10% in Norway [
15], 11–17% in Sweden [
18,
19], to 21% in Denmark [
16]. It should be noted that the more recent seroepidemiologic surveys have utilized the same IFA as in the present study, which is based on a human isolate of
A. phagocytophilum [
16,
17,
19], whereas the older studies have used an equine
A. phagocytophilum isolate [
15,
18]. In contrast to the relatively high seroprevalence of
A. phagocytophilum antibodies, there is a scarcity of case reports of anaplasmosis from the Scandinavian countries [
20,
21]. The main explanation for this disparity is that the European variant of this infectious disease is relatively mild in humans, at least compared to human anaplasmosis in North America [
22]. Presumably, the human-tropic European
A. phagocytophilum strains are less virulent than the American ones and give rise to discrete symptoms or only subclinical infections in the majority of cases. Thus, symptomatic anaplasmosis appears to be a rare disease in Scandinavia. This may account for our inability to detect
A. phagocytophilum DNA in the serum samples derived from the
Anaplasma-reactive anonymous patient samples, even when using two different PCRs targeting different
A. phagocytophilum genes. However, we cannot exclude that our use of serum or plasma may have given a poorer DNA yield compared to if we had used buffy coat or whole blood in view of the intracellular nature of
A. phagocytophilum, which resides within granulocytes.
It is possible that the Anaplasma seroreactivity we have detected among neoehrlichiosis patients reflects previous exposure to A. phagocytophilum. We have no evidence to suggest that the neoehrlichiosis patients were doubly infected with A. phagocytophilum and Ca. N. mikurensis since all patients were negative for A. phagocytophilum by PCR and confirmatory sequencing of the Ca. N. mikurensis PCR amplicons was in no case ambiguous.
The third possible explanation for why up to every fifth of the neoehrlichiosis patients presented with
Anaplasma antibodies is that these antibodies were in fact directed against
Ca. N. mikurensis and cross-reactive with
A. phagocytophilum antigens. One indication that this might be the case is the semblance of an antibody response among the few neoehrlichiosis patients from whom it was possible to obtain repeated blood samples in the present study. Two published studies have also implied that neoehrlichiosis patients may respond with
Anaplasma-reactive antibodies, indicative of cross-reactivity [
2,
6]. However, the issue of cross-reactivity will only be addressable once
Ca. N. mikurensis antigens are available, which will require its cultivation.
Irrespective of the underlying mechanisms behind the
A. phagocytophilum seroreactivity demonstrated by some neoehrlichiosis patients, the main significance of this finding is that a certain degree of vigilance is warranted: patients believed to have anaplasmosis may in fact have neoehrlichiosis. Moreover, patients queried for
A. phagocytophilum antibodies that turn out to be seronegative may have neoehrlichiosis. Two of the neoehrlichiosis patients described in this study were discovered among patient samples submitted for
A. phagocytophilum serology thanks to relevant clinical data, both of whom were seronegative for
Anaplasma. A correct diagnosis is of utmost importance since these two infectious diseases differ with regard to one vital aspect: neoehrlichiosis patients have a substantial risk of contracting vascular events such as deep vein thrombosis, arterial aneurysms or transitory ischemic attacks, which are not recognized to be part of an infectious process [
5]. If neoehrlichiosis patients with vascular complications are correctly diagnosed and adequately treated with antibiotics, they do not incur new vascular events [
5].
To conclude, patients with fever of uncertain origin or with suspected anaplasmosis may in fact have neoehrlichiosis. Misdiagnosed or undiagnosed cases of neoehrlichiosis may be identified either among Anaplasma seropositive patients or among patient samples queried for Anaplasma antibodies that are negative by A. phagocytophilum IFA but have typical risk factors for severe neoehrlichiosis. We recommend that such patients be assayed for the presence of Ca. N. mikurensis DNA by PCR performed on EDTA blood or plasma to determine if they have contracted neoehrlichiosis.
Acknowledgments
Charlotte Sværke-Jørgensen, Head of the Serology Laboratory at Statens Serum Institut, Denmark, is thanked for provision of the relevant serum samples. Anneli Bjoersdorff, AniCura, Sweden, is recognized for provision of positive control samples containing A. phagocytophilum DNA.
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